Stress may aggravate the development of inflammatory bowel disease and irritable bowel syndrome, in which the number of enterochromaffin (EC) cells and 5-hydroxytryptamine (5-HT) levels are abnormal, but the underlying mechanism remains largely unresolved. In this study, we discovered that restraint stress triggered the expression of Tph1, which led to 5-HT production. The 5-HT signaling then increased intestinal permeability, downregulated the expression of tight junction proteins, reduced the number of goblet cells and their ability to secrete mucin, promoted the expression of inflammatory cytokines, and ultimately damaged the intestinal mucosal barrier. Mechanistically, the 5-HT receptor HTR7 was highly expressed in the intestine. It interacted with 5-HT to initiate the Wnt/beta-catenin signaling pathway, inducing an increase in intestinal EC cells and further promoting 5-HT secretion. Additionally, the activation of the Wnt/beta-catenin signaling pathway could initiate the NF-kappaB signaling pathway and induce the expression of inflammatory cytokines. Blocking the 5-HT signal in mice inhibited the activation of the Wnt/beta-catenin signal, thereby alleviating intestinal inflammation. Our findings revealed a novel role for 5-HT in intestinal inflammatory diseases and represent a potential new therapeutic target.